Measuring the Dissociation Constants of Ligands from PMCA Complexes by a Photoactivatable Phosphatidylcholine Membrane Domain Probe

The purpose of this work was to obtain structural information about conformational changes of the plasma membrane Ca2+ pump (PMCA) in the membrane region upon interaction with ATP, Ca2+, calmodulin and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ and ATP under different conditions. With this method we were able to measure apparent and equilibrium constants for the dissociation of Ca2+, ATP and calmodulin and other ligands from PMCA complexes through the change of transmembrane conformations of the pump. The results indicate that the PMCA possesses a high-affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by calmodulin and calmodulin-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. The data further suggest that the hydrophobic transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation. With grants from ANPCYT, CONICET, UBACYT and NIH.